Various fats and fatty oils comprise triglycerides of fatty acids. Many fatty acids, particularly polyunsaturated fatty acids, are difficult to synthesize and can only be obtained by extraction from natural fats or fatty oils in which they naturally occur. Many of these unsaturated fatty acids are known to be valuable for their therapeutic properties. Others are thought to have undesirable effects. Furthermore, the fatty acids having therapeutic properties must be in a particular cis-trans isomeric form. There has long been a need for obtaining individual unsaturated fatty acids in pure form and high quantity, without cis-trans conversion.
Two particular polyunsaturated fatty acids which have been shown to have therapeutic efficacy, and which are difficult to obtain in pure form and large quantities, are (all-Z)-5,8,11,14,17-eicosapentaenoic acid, hereinafter referred to as EPA, and (all-Z)-4,7,10,13,16,19-docosahexaenoic acid, hereafter referred to as DHA. Both EPA and DHA are known to be precursors in the biosynthesis of prostaglandin PGE3.
It is well known that ω-3 fatty acids, such as EPA and DRA, are useful dietary compounds for preventing arteriosclerosis and coronary heart disease, for alleviating inflammatory conditions, and for retarding the growth of tumor cells. Recently, DHA has been the focus of attention because this polyunsaturated fatty acid is the major fatty acid that occurs in the brain and the retina. Also, DHA is the only member of the ω-3 fatty acids that occurs naturally in mother's milk.
British Patents Nos. 1,604,554 and 2,033,745, disclose the use of ω-3 fatty acids for treating thromboembolic conditions. British patent No. 2,148,713, to the present inventor, also discloses the use of EPA and/or DHA, in combination with other fatty acids, for reduction of serum cholesterol and triglyceride levels.
It has heretofore been very difficult to obtain pure EPA and DHA, since the main source of these fatty acids is in the fats and oils of marine animals, fish oils (such as mackerel oil, menhaden oil, salmon oil, capelin oil, tuna oil, sardine oil, or cod oil), marine algae such as Schizochytrium sp., human milk, and vegetable oils, such as linseed oil, either as itself or in the form of a derivative such as a triglyceride. It is difficult to obtain pure DHA or EPA, because these sources normally contain a substantial amount of fatty acid residues, often as residues of triglyceride molecules, which dilute the concentration of DHA or EPA in the oil. Other fatty acids are always present in larger amounts. Since EPA and DHA are known to be medically effective for treating a variety of conditions, highly pure EPA and DHA are required in large amounts to conduct clinical studies and for therapy.
The beneficial effects of ω-3 fatty acids result both from competitive inhibition of compounds produced from ω-6 fatty acids, and from beneficial compounds produced directly from the ω-3 fatty acids themselves. Ω-6 fatty acids are the predominant fatty acids found in plants and animals. Most commercially available ω-3 fatty acids are obtained from certain fish oils, which contain up to 20-30% of these fatty acids. Consequently, large quantities of fish oil are processed and encapsulated each year for sale as a dietary supplement. However, there are several significant problems with these fish oil supplements, including bioaccumulation of fat-soluble vitamins and high levels of saturated and ω-6 fatty acids, both of which can have deleterious health effects.
Previous methods for extracting EPA, DHA and other useful polyunsaturated fatty acids from their triglycerides have not been satisfactory for producing highly pure fatty acids. The term “purity” is used here to mean not only in the sense of being separated from all other fatty acids of different chain lengths, and different number and placement of unsaturation, but also the purity of the particular cis-trans structure. Prior methods not only did not yield sufficient purity, but in many cases also required such extreme physical and chemical conditions as to cause some degree of degradation of the fatty acids, formation of peroxides, and/or conversion of some of the cis- bonds to the trans-form. Furthermore, many prior art processes use materials which are not on the Generally Recognized as Safe (GRAS) list of the U.S. Food and Drug Administration. In order for the final product to be used in foods and drugs, it is important that there be no nonGRAS substances in the final product.
Fujita et al., in U.S. Pat. No. 4,377,526, disclose one method for purifying EPA. In this patent, a mixture of fatty acids containing EPA is treated with urea in order to remove saturated fatty acids and fatty acids of lower unsaturation. The resultant solution is then subjected to fractional distillation in order to obtain higher yields of EPA. The fractional distillation, however, requires a temperature of at least 180° C. over a period of at least 40 minutes. The best purity which can be obtained by this method set forth in any of the examples is 92.9%. Furthermore, it has been discovered that a substantial amount of the EPA produced by this method, in some cases as much as 20%, has some degree of cis-trans conversion. Any amount of the trans-form of EPA is strictly undesirable for food or pharmaceutical use.
Abu-Nasr et al., J. Am. Oil Chemists Soc. 31, 41-45 (1954) disclose isolation of methyl eicosapentaenoate and ethyl docosahexaenoate using cod liver oil acids, using preliminary concentration by precipitation of the pure complexes followed by chromatographic separations. This technique does not give high enough purity, and chromatographic separations require undesirably high amounts of solvent.
Teshima et al., in Bulletin of the Japanese Society of Scientific Fisheries, 44(8) 927 (1978) describe a method for isolating EPA and DHA from squid liver oil by saponifying with ethanolic potassium hydroxide, extracting the fatty acids with ether, and methylating. The crude fatty acid methyl ester is purified by column chromatography on Silica Gel 60, and then the EPA is separated from the DHA by column chromatography on a mixture of silver nitrate and silica gel. The problem with this technique is that there are often traces of silver left in the final product, which is extremely undesirable in a food or pharmaceutical for human consumption. Furthermore, very high amounts of solvent are necessary to conduct the column chromatography.
Other disclosures of the use of column chromatography to separate and purify EPA to some extent are described in Japanese Kokai No. 56, -115736 and Russian No. 973,128.
Rubin, in British Patent No. 2,148,713, describes a process in which the double bonds of the unsaturated fatty acids, in a mixture of fatty acids, are iodinated, followed by saponification of the iodinated oil, extraction of the fatty acids from the saponification mixture, methylation of the iodinated fatty acids, separation of the fatty acids by column chromatography, and then deiodination of the desired fractions. This process permits excellent resolution of the fatty acids upon eventual column chromatography, and protects the fatty acids from oxidation during processing. When used to separate EPA from a natural source of EPA, such as cod liver oil, a yield of over 90% and a purity of 96-100% may be obtained. It has been found, however, that a substantial amount of cis-trans conversion occurs in the course of this process, so that the product obtained is not pure all-cis EPA. Furthermore, iodine is not on the list of GRAS materials.
Rubin, in U.S. Pat. No. 4,792,418, describes a process for obtaining pure polyunsaturated fatty acids such as EPA and DHA and their esters, without degradation thereof. This process involves first hydrolyzing the triglycerides of the oil source under mild conditions, as with lipase, removing non-saponifiable material by washing with organic solvent, treating with urea in order to remove saturated and monounsaturated fatty acids to form a urea complex with saturated and mono-saturated fatty acids, dissolving the remainder in an organic solvent, preferably acetone, slowly cooling and fractionally removing solidified material as it forms.
Barclay, in U.S. Pat. Nos. 5,518,918, and 6,451,567, discloses production of ω-3 fatty acids by growing Thraustochytrium, Schizochytrium, and mixtures thereof with high ω-e3 fatty acid content in a fermentation medium. However, this process produces a mixture of ω-3 fatty acids, rather than individual ω-3 fatty acids.
Best et al., in U.S. Pat. No. 5,928,696, extract oils from native substances using centrifugation. Again, this method produces mixtures of unsaturated fatty acids rather than pure individual fatty acids.
Kyle et al., in U.S. Pat. No. 5,397,591, disclose a method for obtaining DHA from cultivation of dinoflagellates in a fermentor, induction of the dinoflagellates to produce single cell oil having a high proportion of DHA, and recovery of that oil. Preferably, the oil recovered contains at least about 20% by weight of DHA, and more preferably, more than about 35% by weight DHA. The product recovered is not pure DHA, but a mixture of DHA in other oils.
Cornieri et al., in U.S. Pat. No. 5,130,061, disclose a process for extracting polyunsaturated fatty acid esters from fish oils. However, the product obtained is a mixture of EPA and DHA esters.